Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Other methods or instruments for diagnosis, e.g. instruments for taking a cell sample, for biopsy, for vaccination diagnosis; Sex determination; Ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B10/007—Devices for taking samples of body liquids for taking urine samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

• the present disclosure relates to the use of the presence or absence or amplitude of one or more peptide markers in a sample from a subject for the (differential) diagnosis of bladder cancer and to a method for the diagnosis of bladder cancer, wherein the presence or absence or amplitude of the peptide marker or markers is indicative of the existence and of the tumor stage of bladder cancer.
• Bladder cancer is a malignant tumor on the mucous membrane of the bladder. Bladder cancer is one of the most common malignant diseases. In the urological field, it is the second most frequent cancer disease after prostate cancer. In the German-speaking area, there is an incidence of about 22 of 100,000 humans per year. In males, bladder cancer occurs about twice to three times as frequent as in females. Every year, an estimated 13,000 males and 5,000 females become afflicted with the disease in the Federal Republic of Germany. Bladder cancer is a disease of advanced age. The disease risk increases from the 40th year with increasing age.
• bladder cancer can be detected earlier thereby. If there is a suspicion of a neoplasm in the bladder, for example, if blood was observed in the urine or if there are continuing symptoms of bladder irritation, a cystoscopy is performed. When the examiner sees a tumor in the bladder wall, they can estimate which wall layers are penetrated by the tumor, and they can also take samples, which are then examined macroscopically. Depending on the tumor growth, a distinction is made between superficial and infiltrating (tissue-entering) carcinomas.
• the latter have already grown into the muscles of the bladder and can spread into the neighboring organs (for example, the prostate gland in males or the uterus in females).
• the histological-pathological classification of the mucous membrane tumors is effected according to the TNM system.
• pTa non-invasive papillary carcinoma of the mucous membrane (urothelium)
• pTcis carcinoma in situ
• pT1 infiltration below the mucous membrane (subepithelial connective tissue), subclassification pT1a-c
• a radiological examination of the whole urinary tract can be performed.
• the urine is examined for malignant cells under a microscope.
• further diagnostic methods are employed, such as sonography, CT (computer tomography) or MRT (magnetic resonance tomography).
• sonography computer tomography
• MRT magnetic resonance tomography
• the present disclosure relates to the use of the presence or absence or amplitude of at least one polypeptide marker in a sample from a subject for the diagnosis of bladder cancer, wherein said polypeptide marker is selected from polypeptide marker Nos. 1 to 836 as characterized by the molecular masses and migration times as stated in Table 1.
• the present disclosure it is possible to diagnose bladder cancer at a very early stage.
• the disease can be cured by known methods at an early stage.
• the disclosure further enables an inexpensive, quick and reliable evaluation of the tumor stage and a diagnosis with in part non-invasive or only minimal-invasive operations.
• the migration time is determined by capillary electrophoresis (CE), for example, as set forth in the Example under item 2.
• CE capillary electrophoresis
• a glass capillary of 90 cm in length and with an inner diameter (ID) of 50 ⁇ m and an outer diameter (OD) of 360 ⁇ m is operated at an applied voltage of 30 kV.
• the mobile solvent for example, 300% methanol, 0.5% formic acid in water is used.
• CE migration times may vary. Nevertheless, the order in which the polypeptide markers are eluted is typically the same under the stated conditions for any CE system employed. In order to balance any differences in the migration time that may nevertheless occur, the system can be normalized using standards for which the migration times are exactly known. These standards may be, for example, the polypeptides stated in the Examples (see the Example, item 3).
• the characterization of the polypeptides shown in Tables 1 to 8 was determined by means of capillary electrophoresis-mass spectrometry (CE-MS), a method which has been described in detail, for example, by Neuhoff et al. (Rapid communications in mass spectrometry, 2004, Vol. 20, pages 149-156).
• CE-MS capillary electrophoresis-mass spectrometry
• the variation of the molecular masses between individual measurements or between different mass spectrometers is relatively small when the calibration is exact, typically within a range of ⁇ 0.10%, preferably within a range of ⁇ 0.050%, more preferably ⁇ 0.030%.
• polypeptide markers are proteins or peptides or degradation products of proteins or peptides. They may be chemically modified, for example, by posttranslational modifications, such as glycosylation, phosphorylation, alkylation or disulfide bridges, or by other reactions, for example, within the scope of degradation. In addition, the polypeptide markers may also be chemically altered, for example, oxidized, during the purification of the samples.
• the polypeptides according to the disclosure are used to diagnose bladder cancer, on the one hand, and to enable a distinction between different tumor stages, on the other.
• “Diagnosis” means the process of knowledge gaining by assigning symptoms or phenomena to a disease or injury.
• the existence of bladder cancer and the tumor stage of the bladder carcinoma is concluded from the presence or absence or amplitude of particular polypeptide markers.
• the polypeptide markers according to the disclosure are determined in a sample from a subject, wherein their presence or absence allows to conclude the existence of bladder cancer in the case of frequency markers, or the difference in signal intensities allows to conclude the existence of bladder cancer in the case of amplitude markers.
• the presence or absence or amplitude of a polypeptide marker can be measured by any method known in the prior art. Methods which may be used are exemplified below.
• a polypeptide marker is considered present if its measured value is at least as high as its threshold value. If the measured value is lower, then the polypeptide marker is considered absent.
• the threshold value can be determined either by the sensitivity of the measuring method (detection limit) or defined from experience.
• the threshold value is considered to be exceeded preferably if the measured value of the sample for a certain molecular mass is at least twice as high as that of a blank sample (for example, only buffer or solvent).
• the polypeptide marker or markers is/are used in such a way that its/their presence or absence is measured, wherein the presence or absence is indicative of bladder cancer (frequency markers).
• bladder cancer frequency markers
• polypeptide markers which are typically present in patients with bladder cancer (ill) such as polypeptide markers No. 1 to 44, but absent or only rarely present in subjects with no bladder cancer (control).
• polypeptide markers which are present in subjects with no bladder cancer, but are less frequently or not at all present in subjects with bladder cancer for example, Nos. 45 to 149 (Table 2).
• a frequency marker is a variant of an amplitude marker in which the amplitude is low in some samples. It is possible to convert such frequency markers to amplitude markers by including the corresponding samples in which the marker is not found into the calculation of the amplitude with a very small amplitude, on the order of the detection limit.
• Occurrence in Occurrence in Number group BC Control 1 0.86 0.13 2 0.71 0 3 0.73 0.03 4 0.71 0.07 5 0.64 0.03 6 0.64 0.03 7 0.71 0.13 8 0.71 0.13 9 0.73 0.16 10 0.67 0.09 11 0.57 0 12 0.92 0.35 13 0.57 0 14 0.57 0 15 0.57 0.03 16 0.57 0.03 17 0.57 0.03 18 0.57 0.03 19 0.57 0.03 20 0.67 0.13 21 0.78 0.25 22 0.69 0.16 23 0.78 0.25 24 0.69 0.17 25 0.56 0.03 26 0.57 0.05 27 0.67 0.16 28 0.6 0.09 29 0.51 0 30 0.86 0.35 31 0.91 0.41 32 0.71 0.22 33 0.51 0.03 34 0.58 0.09 35 0.89 0.41 36 0.51 0.03 37 0.73 0.25 38 0.51 0.03 39 0.62 0.16 40 0.69 0.22 41 0.58 0.13 42 0.58 0.13 43 0.73 0.38 44 0.8 0.5 45 0.42 0.72 46 0.51 0.81 47 0.44 0.75 48 0.62
• markers 1-146 are particularly preferred.
• the amplitude markers as stated in Tables 3 and 4 may also be used for the diagnosis of bladder cancer (Nos. 150-836). Amplitude markers are used in such a way that the presence or absence is not critical, but the height of the signal (the amplitude) decides if the signal is present in both groups.
• Tables 3 and 4 the mean normalized amplitudes of the corresponding signals (characterized by mass and migration time) averaged over all samples measured are stated. Two normalization methods are possible to achieve comparability between differently concentrated samples or different measuring methods.
• markers 150-185 is particularly preferred.
• All groups employed consist of at least 40 individual patient or control samples in order to obtain a reliable mean amplitude.
• the decision for a diagnosis is made as a function of how high the amplitude of the respective polypeptide markers in the patient sample is in comparison with the mean amplitudes in the control groups or the bladder cancer group. If the amplitude rather corresponds to the mean amplitudes of the bladder cancer group, the existence of bladder cancer is to be considered, and if it rather corresponds to the mean amplitudes of the control group, the non-existence of bladder cancer is to be considered.
• the distance between the measured value and the mean amplitude can be considered a probability of the sample's belonging to a certain group.
• the polypeptide markers are also suitable for determining a tumor stage.
• the frequencies according to Table 5 and the amplitudes according to Table 6 are suitable.
• the markers according to the disclosure can also be employed for the differential diagnosis of the individual stages pTa to pT4.
• the subject from which the sample in which the presence or absence or the amplitude of one or more polypeptide markers is determined is derived may be any subject which is capable of suffering from bladder cancer, for example, an animal or human.
• the subject is a mammal, and most preferably, it is a human.
• not just one polypeptide marker but a combination of markers are used to diagnose bladder cancer, wherein the existence of bladder cancer is concluded from their presence or absence and/or the height of the amplitude.
• a bias in the overall result from a few individual deviations from the typical presence probability in the sick or control individual can be reduced or avoided.
• the sample in which the presence or absence or amplitude of the polypeptide marker or markers according to the disclosure is measured may be any sample which is obtained from the body of the subject.
• the sample is a sample which has a polypeptide composition suitable for providing information about the state of the subject (bladder cancer or not).
• it may be blood, urine, synovial fluid, tissue fluid, a body secretion, sweat, cerebrospinal fluid, lymph, intestinal, gastric, pancreatic juices, bile, lachrymal fluid, a tissue sample, sperm, vaginal fluid or a faeces specimen.
• it is a liquid sample.
• the sample is a urine sample or blood sample
• the sample is a urine sample or blood sample, wherein said blood sample may be a (blood) serum or (blood) plasma sample.
• Urine samples can be taken as known in the prior art.
• a midstream urine sample is used in the context of the present disclosure.
• the urine sample may be taken by means of a catheter or also by means of an urination apparatus as described in WO 01/74275.
• Blood samples can be taken by methods known in the prior art, for example, from a vein, artery or capillary.
• a blood sample is obtained by withdrawing venous blood by means of a syringe, for example, from an arm of the subject.
• the term “blood sample” includes samples obtained from blood by further purification and separation methods, such as blood plasma or blood serum.
• the presence or absence of a polypeptide marker in the sample may be determined by any method known in the prior art that is suitable for measuring polypeptide markers. Such methods are known to the skilled person. In principle, the presence or absence of a polypeptide marker can be determined by direct methods, such as mass spectrometry, or indirect methods, for example, by means of ligands.
• the sample from the subject may be pretreated by any suitable means and, for example, purified or separated before the presence or absence of the polypeptide marker or markers is measured.
• the treatment may comprise, for example, purification, separation, dilution or concentration.
• the methods may be, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods, such as affinity separation or separation by means of ion-exchange chromatography, or electrophoretic separation.
• Particular examples thereof are gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary electrophoresis, metal affinity chromatography, immobilized metal affinity chromatography (IMAC), lectin-based affinity chromatography, liquid chromatography, high-performance liquid chromatography (HPLC), normal and reverse-phase HPLC, cation-exchange chromatography and selective binding to surfaces. All these methods are well known to the skilled person, and the skilled person will be able to select the method as a function of the sample employed and the method for determining the presence or absence of the polypeptide marker or markers.
• the sample, before being measured is separated by capillary electrophoresis, purified by ultracentrifugation and/or divided by ultrafiltration into fractions which contain polypeptide markers of a particular molecular size.
• a mass-spectrometric method is used to determine the presence or absence of a polypeptide marker, wherein a purification or separation of the sample may be performed upstream from such method.
• mass-spectrometric analysis has the advantage that the concentration of many (>100) polypeptides of a sample can be determined by a single analysis. Any type of mass spectrometer may be employed. By means of mass spectrometry, it is possible to measure 10 fmol of a polypeptide marker, i.e., 0.1 ng of a 10 kDa protein, as a matter of routine with a measuring accuracy of about ⁇ 0.01% in a complex mixture.
• an ion-forming unit is coupled with a suitable analytic device.
• electrospray-ionization (ESI) interfaces are mostly used to measure ions in liquid samples, whereas the matrix-assisted laser desorption/ionization (MALDI) technique is used for measuring ions from a sample crystallized with a matrix.
• MALDI matrix-assisted laser desorption/ionization
• quadrupoles, ion traps or time-of-flight (TOF) analyzers may be used.
• electrospray ionization the molecules present in solution are atomized, inter alia, under the influence of high voltage (e.g., 1-8 kV), which forms charged droplets that become smaller from the evaporation of the solvent.
• high voltage e.g. 1-8 kV
• Coulomb explosions cause the formation of free ions, which can then be analyzed and detected.
• gas-phase ion spectrometry such as laser desorption/ionization mass spectrometry, MALDI-TOF MS, SELDI-TOF MS (surface-enhanced laser desorption/ionization), LC-MS (liquid chromatography/mass spectrometry), 2D-PAGE/MS and capillary electrophoresis-mass spectrometry (CE-MS). All methods mentioned are known to the skilled person.
• CE-MS in which capillary electrophoresis is coupled with mass spectrometry. This method has been described in some detail, for example, in the German Patent Application DE 10021737, in Kaiser et al. (J Chromatogr A, 2003, Vol. 1013: 157-171, and Electrophoresis, 2004, 25: 2044-2055) and in Wittke et al. (Journal of Chromatography A, 2003, 1013: 173-181).
• the CE-MS technology allows to determine the presence of some hundreds of polypeptide markers of a sample simultaneously within a short time and in a small volume with high sensitivity. After a sample has been measured, a pattern of the measured polypeptide markers is prepared.
• This pattern can be compared with reference patterns of sick or healthy subjects. In most cases, it is sufficient to use a limited number of polypeptide markers for the diagnosis of bladder cancer and the differential diagnosis between different stages of bladder cancer, for example, at least 6, 8, 10, 20, 50 or 100 markers.
• a CE-MS method which includes CE coupled on-line to an ESI-TOF MS device is further preferred.
• solvents for CE-MS, the use of volatile solvents is preferred, and it is best to work under essentially salt-free conditions.
• suitable solvents include acetonitrile, methanol and the like.
• the solvents can be diluted with water or admixed with a weak acid (e.g., from 0.10% to 1% formic acid) in order to protonate the analyte, preferably the polypeptides.
• capillary electrophoresis By means of capillary electrophoresis, it is possible to separate molecules by their charge and size. Neutral particles will migrate at the speed of the electroosmotic flow upon application of a current, while cations are accelerated towards the cathode, and anions are delayed.
• the advantage of capillaries in electrophoresis resides in their favorable ratio of surface to volume, which enables a good dissipation of the Joule heat generated during the current flow. This in turn allows high voltages (usually up to 30 kV) to be applied and thus a high separating performance and short times of analysis.
• silica glass capillaries having inner diameters of typically from 50 to 75 ⁇ m are usually employed. The lengths employed are from 30 to 100 cm.
• the capillaries are usually made of plastic-coated silica glass.
• the capillaries may be both untreated, i.e., expose their hydrophilic groups on the interior surface, or coated on the interior surface.
• a hydrophobic coating (coating: a method that conceals the negative polarized surface of silica, for example) may be used to improve the resolution.
• a pressure may also be applied, which typically is within a range of from 0 to 1 psi. The pressure may also be applied only during the performance or altered meanwhile.
• the markers of the sample are separated by means of capillary electrophoresis, then directly ionized and transferred on-line to a mass spectrometer coupled thereto for detection.
• polypeptide markers for the diagnosis of bladder cancer.
• at least three polypeptide markers may be used, for example, markers 1, 2 and 3; 1, 2 and 4; etc.
• Urine was withdrawn from healthy donors (control group) as well as from patients suffering from bladder cancer.
• 700 ⁇ l of urine was removed and admixed with 700 ⁇ l of filtration buffer (2 M urea, 10 mM ammonia, 0.02% SDS).
• This 1.4 ml of sample volume was ultrafiltrated (20 kDa, Sartorius, Göttingen, Germany). The ultrafiltration was performed at 3000 rpm in a centrifuge until 1.1 ml of ultrafiltrate was obtained.
• CE-MS measurements were performed with a capillary electrophoresis system from Beckman Coulter (P/ACE MDQ System; Beckman Coulter Inc., Fullerton, USA) and an ESI-TOF mass spectrometer from Bruker (micro-TOF MS, Bruker Daltonik, Bremen, Germany).
• the CE capillaries were supplied by Beckman Coulter and had an ID/OD of 50/360 ⁇ m and a length of 90 cm.
• the mobile phase for the CE separation consisted of 20% acetonitrile and 0.25% formic acid in water.
• For the “sheath flow” on the MS 300% isopropanol with 0.50% formic acid was used at a flow rate of 2 ⁇ l/min.
• the coupling of CE and MS was realized by a CE-ESI-MS Sprayer Kit (Agilent Technologies, Waldbronn, Germany).
• a pressure of from 1 to a maximum of 6 psi was applied, and the duration of the injection was 99 seconds.
• about 150 to 900 nl of the sample was injected into the capillary, which corresponds to about 100% to 500% of the capillary volume.
• a stacking technique was used to concentrate the sample in the capillary.
• a 1 M NH 3 solution was injected for 7 seconds (at 1 psi)
• a 2 M formic acid solution was injected for 5 seconds.
• the separation voltage (30 kV) was applied, the analytes were automatically concentrated between these solutions.
• the subsequent CE separation was performed with a pressure method: 40 minutes at 0 psi, then 0.1 psi for 2 min, 0.2 psi for 2 min, 0.3 psi for 2 min, 0.4 psi for 2 min, and finally 0.5 psi for 32 min.
• the total duration of a separation run was thus 80 minutes.
• the nebulizer gas was set to the lowest possible value.
• the voltage applied to the spray needle for generating the electrospray was 3700-4100 V.
• the remaining settings at the mass spectrometer were optimized for peptide detection according to the manufacturer's protocol. The spectra were recorded over a mass range of m/z 400 to m/z 3000 and accumulated every 3 seconds.
• Protein/polypeptide Migration time Aprotinin (SIGMA, Tauf Wegn, DE, Cat. # A1153) 9.2 min Ribonuclease (SIGMA, Tauf Wegn, DE, Cat. # R4875) 10.9 min Lysozyme (SIGMA, Tauf Wegn, DE, Cat.

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